KR20130005208A - Method and kit for detecting carbapenem resistant enterobacteriaceae using real-time pcr - Google Patents

Abstract

PURPOSE: A method and a kit for detecting carbapenem resistant Enterobacteriaceae using real-time PCR are provided to quickly and accurately detect Enterobacteriaceae infection from a test sample of a patient who is suspected to have Enterobacteriaceae infection. CONSTITUTION: A method for detecting carbapenem resistant Enterobacteriaceae using real-time PCR comprises: a step of preparing a gene sample from a test sample; a step of amplifying a gene which is specific to carbapenem resistant Enterobacteriaceae in the gene sample using a primer pair and a marker by real-time PCR; a step of measuring Enterobacteriaceae infection degree in the test sample; and a step of determining Enterobacteriaceae infection. The primer pair includes: a primer pair of sequence numbers 1 and 2 for KPC gene; a primer pair of sequence numbers 3 and 4 for VIM gene; a primer pair of sequence numbers 5 and 6 for NDM gene; a primer pair of sequence numbers 7 and 8 for GES gene; a primer pair of sequence numbers 9 and 10 for IMP(-1,-3,-6,-10) gene; a primer pair of sequence numbers 11 and 12 for IMP(-4,-26) gene; or a primer pair of sequence numbers 13 and 14 for IMP(-2,-8,-19,-20,-24) gene which is specific to carbapenem resistant Enterobacteriaceae. [Reference numerals] (AA) KPC sensitivity test; (BB) VIM sensitivity test; (CC) NDM sensitivity test; (DD) GES sensitivity test; (EE) IMP(1,3,4,6,10,26) sensitivity test; (FF) IMP(2,8,19,20,24) sensitivity test

Description

Method and kit for detecting carbapenem resistant enterobacteriaceae using real-time PCR}

The present invention relates to a method and kit for rapidly and accurately examining whether a carbapenem-resistant enterococci is present in a sample using real-time PCR (real-time polymerase chain reaction), and more particularly, to a patient suspected of carbapenem-resistant enterobacteriaceae infection. The present invention relates to a method and kit for quickly and accurately testing for early infection of carbapenem-resistant enterococci from a specimen.

More specifically, the present invention uses real-time PCR to detect high-specificity and sensitivity of carbapenem-resistant enterobacteria, which may be present in a sample of suspected carbapenem-resistant enterobacteriaceae infection, based on rapid, accurate and quantified data, Early detection of small amounts of carbapenem-resistant enterobacteria in the early stages of infection with penem-resistant enterococci enables the proper dosing treatment at the beginning of the infection, preventing the patient's treatment time and preventing the spread of infection in advance. It relates to methods and kits.

The carbapenem family of antibiotics is an antibiotic widely used for the treatment of Gram-negative bacterial infections. Carbapenem family of antibiotics include imipenem, meropenem, Invanz, ertapenem, and doripenem. Currently, the carbapenem family of antibiotics is known to be the most potent antibiotic and is mainly used in patients with severe infection.

However, due to the indiscriminate use of antibiotics, bacteria that are resistant to various antibiotics have arisen and are a social problem. The antibiotics of the carbapenem family are also becoming socially problematic as resistant strains are reported worldwide. Intestinal bacteria that are resistant to these carbapenem family of antibiotics are called carbapenem resistant enterobacteriaceae (CRE).

Enterobacteriaceae resistant to the carbapenem family of antibiotics are known to neutralize the carbapenem family of antibiotics by producing an enzyme called β-lactamase.

On the other hand, antibiotics can be divided into four major mechanisms. First, there is a mechanism of antibacterial action by inhibiting the cell wall synthesis of bacteria, second, there is a mechanism of antibacterial action by inhibiting the protein synthesis of bacteria. Third, there is a case of inhibiting the synthesis of nucleic acid (DNA, RNA) of bacteria to have an antimicrobial effect, and finally the mechanism of antimicrobial action by inhibiting the metabolic pathway of bacteria.

In the case of the carbapenem antibiotic, it has an antimicrobial effect as a mechanism for inhibiting or inhibiting the cell wall synthesis of bacteria. Intestinal bacteria that are resistant to the carbapenem family of antibiotics produce an enzyme called β-lactamase, which induces enzyme inactivation by the enzyme, resulting in antibiotic resistance.

The bacterium that is resistant to the carbapenem family of antibiotics is E. coli and Klebsiella pneumoniae , and other strains with genes capable of producing β-lactamase enzymes. It is known to have.

As genes for generating β-lactamase, genes such as KPC, IMP, GES, SIM, and VIM have been reported, including the NDM gene. Genes producing these β-lactamase enzymes exist in several genotypes. The most representative New Delhi metallo beta lactamase (NDM) was named after 2008 when Swedish patients were confirmed to have been infected during surgery in New Delhi, India. Bacteria that carry the NDM gene are known to be resistant to almost all existing antibiotics, and infection experts are concerned that they have recently been found in India and Pakistan and subsequently found in the United States and Japan. As the risk of multidrug-resistant bacteria including CRE emerged worldwide, Korea also urgently designated 'cabapenem-resistant enterobacterial (CRE)' as a statutory epidemic in September 2010.

Infection with carbapenem-resistant enterobacteria is known to cause diseases such as high fever, pneumonia and urethritis. In particular, patients undergoing long-term chemotherapy, children, or elderly people with weakened immune function, such as the elderly are very fatal, and if infected will lead to sepsis and shock death.

However, until now, the standard of treatment for patients infected with carbapenem-resistant enterobacteria has not been determined. Therefore, it is best to determine whether the infection is early and to prevent further spread of infection. Nevertheless, the trend of domestic carbapenem-resistant enterobacteriaceae is increasing.

Therefore, there is a need in the technical field to which the present invention is directed to provide a method for quickly and accurately testing carbapenem-resistant enterobacteriaceae infection and transmission in advance.

In general, the methods of screening for carbapenem-resistant Enterobacteriaceae include disk diffusion, Modified Hodge Test (MHT), Cabapenemase inhibition test, PCR amplification and electrophoresis, and DNA sequencing methods. Used.

In this regard, the Korean Utility Model Registration No. 20-0325376 is a measuring instrument used to determine the sensitivity, tolerance, and intermediate for each antibiotic by measuring the inhibitor band in millimeter units in the antibiotic sensitivity test of microorganisms using the principle of disk diffusion in plate media. It is starting. However, such a conventional method for testing the infection of carbapenem-resistant enterobacteria by using the disk diffusion method has low sensitivity caused by technical errors such as the type of culture medium used and the dilution ratio, ambiguity of reading of test results, and above all, test results. There is a shortcoming of the delay, there is a limit in determining whether the infection of the carbapenem-resistant enterobacterial infection quickly and accurately, there is a limit that can not block the risk of spreading the infection early.

In addition, the DNA sequencing test can only test one specimen with one or two tests, although the results are accurate, it is not only expensive but also time-consuming to test for carbapenem resistance. It is not suitable as a screening method for carbapenem-resistant enterococci on the level of prevention because it does not prevent the intestinal bacteria propagation problem.

On the other hand, the Republic of Korea Patent No. 10-0440746 discloses a microarray DNA chip for screening antibiotic resistant bacteria, the Republic of Korea Patent No. 10-1038519 is a microarray method for testing 64 human infectious diseases pathogens A DNA chip is disclosed.

This microarray DNA chip can simultaneously test tens or tens of thousands of genes on a single slide, or end point analysis that hybridizes the product after PCR amplification to a DNA chip integrated with a probe and simply identifies the genotype. end-point analysis). However, the PCR amplification process is a linear step in which the amplification rate begins to decrease as the amount of DNA amplification begins to gradually deplete after exponential phases where the DNA amplification rate is constant and highly reproducible. (linear phases) and the time when PCR amplification stops, proceed to the plateau phases with the lowest reproducibility. For this reason, the biggest problem of the microarray DNA chip method is that it is difficult to design primers and probes for PCR amplification that can detect a plurality of target genes while satisfying both specificity and sensitivity, and to cut off the fluorescence intensity for positive and negative determination. It is difficult to decide.

In particular, in the microarray DNA chip method, amplification is not performed on some target gene regions according to primer design conditions for PCR amplification of a target gene. As the number increases, a false positive problem occurs due to nonspecific hybridization of primers. That is, when there are multiple target genes and multiple genotypes of each target gene, the design of multiple primers for precisely amplifying them is a trade-off relationship in terms of sensitivity and specificity, so sensitivity and specificity It is difficult to provide a primer that satisfies all of them, and therefore, it is difficult to manufacture a multiplex type microarray DNA chip.

In view of the above problems, the technical field of the present invention is based on the rapid, accurate and quantitative data on whether the carbapenem-resistant enterobacteriaceae is infected in place of the conventional disk diffusion method, DNA sequencing method, and multiplex microarray DNA chip. There is an urgent need to develop a method capable of high specificity and sensitivity.

Accordingly, the present inventors can detect NDM, KPC, IMP, GES and VIM, which are target genes for carbapenem-resistant enterobacteriaceae, by using a rapid and accurate multiplex real time PCR (Multiplex Real Time PCR) technology. It has come to devise methods and kits that can detect multiple genotypes simultaneously based on rapid, accurate and quantified data.

Republic of Korea Utility Model Registration No. 20-0325376 (2003.9.4) Republic of Korea Patent Publication No. 10-1038519 (2011.6.2) Republic of Korea Patent Publication No. 10-0440746 (2004.7.23)

It is an object of the present invention to provide a method and kit for quickly and accurately testing whether a carbapenem-resistant enterobacteriaceae is infected from a sample of a suspected patient with carbapenem-resistant enterobacteriaceae using real-time PCR (real-time polymerase chain reaction). .

In addition, the present invention uses real-time PCR to detect high-specificity and sensitivity of carbapenem-resistant enterobacteria, which may be present in a sample of a suspected carbapenem-resistant enterobacteriaceae infection, on the basis of rapid, accurate and quantified data, and carbapenem resistance. Early detection of the presence of small amounts of carbapenem-resistant enterobacteria in the early stages of enterococci infection, enabling the appropriate dosing treatment at the beginning of the infection to prevent the patient's treatment time or prevent the spread of infection in advance, and The purpose is to provide a kit.

As a result of resolving the technical problem of the present invention and in accordance with the object of the invention, the present inventors have based on the base sequence of NDM, KPC, IMP, GES and VIM genes possessed by carbapenem-resistant enterobacteriaceae. Thus, genotypes of each target gene were compared and analyzed with each other, and using a common nucleotide sequence, one primer set for each target gene was used to detect one or more genotypes.

In addition, in addition to primers that can amplify a specific portion of each target gene, probes complementary to specific sequences of the target gene can be used to produce more accurate results. In particular, the result readout is multiplex real-time PCR. Accurate reading as a numerical value of the Ct value (or Cp value) using the technique was improved to improve the ambiguity result reading of the conventional disk diffusion method as described above. In addition, internal control was added to evaluate the validity of the assay, and two target genes in one test tube were simultaneously used with two probes labeled with markers that could be detected at different wavelengths. The test time can be shortened by allowing to detect all at once.

Carbapenem-resistant enterococci test method using real-time PCR of an embodiment of the present invention,

Preparing a genetic sample from the sample,

Primer pairs of SEQ ID NOS: 1 and 2 for the KPC gene specific for carbapenem-resistant enterobacteria, primer pairs of SEQ ID NOs: 3 and 4 for the VIM gene specific for carbapenem-resistant enterobacteria, using the prepared gene sample as a template, Primer pairs of SEQ ID NOs: 5 and 6 for NDM genes specific for carbapenem-resistant enterobacteria, primer pairs of SEQ ID NOs: 7 and 8 for GES genes specific for carbapenem-resistant enteric bacteria, and IMP (- Primer pairs of SEQ ID NOs: 9 and 10 for 1, -3, -6, -10) genes, primer pairs of SEQ ID NOs: 11 and 12 for IMP (-4, -26) genes specific for carbapenem-resistant enterobacteriaceae, And amplifying at least one primer pair selected from the group consisting of primer pairs of SEQ ID NOs: 13 and 14 for genes of IMP (-2, -8, -19, -20, -24) specific for carbapenem-resistant enterobacteriaceae; Be And cover penem step of tolerance in relation to the specific genes to jangnaegyun using a marker that indicates the gene jeungpokryang, amplified to cover penem-resistant PCR in real time (Real-time) the specific gene to jangnaegyun in the gene sample in the sample,

After amplifying a gene specific for the carbapenem-resistant enterobacteriaceae, measuring the degree of infection of the carbapenem-resistant enterococci in the sample based on the amount of gene amplification specific to the carbapenem-resistant enterobacteria using the labeling substance;

And determining whether the carbapenem-resistant enterobacteriaceae infection is present in the sample based on the measured degree of carbapenem-resistant enterobacteriaceae infection.

In the method for testing carbapenem-resistant enterobacteriaceae using real-time PCR according to an embodiment of the present invention, the labeling substance is a fluorescent substance, and the amplification amount of a gene specific for carbapenem-resistant enterobacteria is determined by measuring the intensity of fluorescence from the labeling substance. Can be calculated.

Carbapenem-resistant enterococci test method using real-time PCR of an embodiment of the present invention,

Before amplification, a positive standard, which is a gene sample that knows the copy number of a gene specific for carbapenem-resistant enterobacteria, is a primer pair corresponding to a gene specific for the carbapenem-resistant enterobacteria, and a gene specific for the amplified carbapenem-resistant enterobacteria. Amplifying by real-time PCR using a labeling material representing the amount of gene amplification in relation to

Measuring a change in fluorescence intensity of the labeling substance over the entire PCR amplification period of the positive standard, and analyzing the number of PCR reactions (Cp value or Ct value) attaining a constant fluorescence intensity;

Matching the copy number of the positive standard with a Cp value or a Ct value from a standard curve obtained by measuring a change in fluorescence intensity of the labeling substance;

The change in fluorescence intensity of the labeling substance over the entire PCR amplification period of the gene specific for the carbapenem-resistant enterobacteriaceae present in the sample of the sample was measured, and the number of PCR reactions (Cp value or Ct that reached a constant fluorescence intensity) was measured. Value),

The degree of infection of carbapenem-resistant enterococci present in the sample by mapping the Cp or Ct values obtained by PCR amplifying the gene specific for the carbapenem-resistant enterobacteria present in the sample of the sample to the copy number of the positive standard. It further comprises the step of measuring quantitatively.

In a method for testing carbapenem-resistant enterobacteriaceae using real-time PCR of an embodiment of the present invention, the labeling substance is an oligo having a nucleotide sequence of SEQ ID NO: 15 as a probe that specifically binds to a KPC gene specific for carbapenem-resistant enterobacteriaceae. A probe consisting of a nucleotide or an oligonucleotide having a nucleotide sequence complementary to such an oligonucleotide, a probe specifically binding to a VIM gene specific for carbapenem-resistant enterobacteriaceae, or an oligonucleotide having a nucleotide sequence of SEQ ID NO: 16 or to such oligonucleotide A probe consisting of oligonucleotides having complementary nucleotide sequences, a brob that binds specifically to an NDM gene specific for carbapenem-resistant enterobacteria, and an oligonucleotide having a nucleotide sequence of SEQ ID NO: 17 or to such oligonucleotide Probes consisting of oligonucleotides with complementary nucleotide sequences, probes that specifically bind to GES genes specific for carbapenem-resistant enterobacteriaceae, oligonucleotides having the nucleotide sequence of SEQ ID NO: 18, or bases complementary to such oligonucleotides Probes consisting of oligonucleotides, probes that specifically bind to IMP (-1, -3, -6, -10) genes specific for carbapenem-resistant enterobacteria, oligonucleotides having the nucleotide sequence of SEQ ID NO: 19 or such oligonucleotides A probe consisting of an oligonucleotide having a nucleotide sequence complementary to that, a probe specifically binding to an IMP (-4, -26) gene specific for carbapenem-resistant enterobacteriaceae, or an oligonucleotide having a nucleotide sequence of SEQ ID NO: 20 or such an oligo; Nucleotide Complementary to Nucleotide A probe consisting of an oligonucleotide having a nucleotide sequence of SEQ ID NO: 21 as a probe specifically binding to a gene of IMP (-2, -8, -19, -20, -24) specific for carbapenem-resistant enterobacteriaceae. It may be labeled at one end of the at least one probe selected from the group consisting of oligonucleotides or probes consisting of oligonucleotides having base sequences complementary to such oligonucleotides, for example at the 5 ′ end.

In this regard, it is preferable to simultaneously use two probes labeled with a label that can be detected at different wavelengths, and one test using two probes labeled with a label that can be detected at different wavelengths simultaneously. More preferably, the tube can detect two target genes specific for carbapenem-resistant enterobacteria at once.

As an example, one end of one probe, eg, 5 'end, is a label that can be detected at 470 nm, and one end of another probe, eg, 5' end, is a label that can be detected at 530 nm. It can be labeled with a substance. For example, the label that can be detected at 470nm may be FAM, and the label that can be detected at 530nm may be VIC. However, the present invention is not limited thereto, and a label having various detection wavelengths may be used.

The real-time PCR amplification kit for detecting carbapenem-resistant enterobacteria of one embodiment of the present invention is a primer pair of SEQ ID Nos. 1 and 2 for a KPC gene specific for carbapenem-resistant enterobacteriaceae, a VIM gene specific for carbapenem-resistant enterobacteriaceae. Primer pairs of SEQ ID NOs: 3 and 4, primer pairs of SEQ ID NOs: 5 and 6 for NDM genes specific for carbapenem-resistant enterobacteria, primer pairs of SEQ ID NOs: 7 and 8 for GES genes specific for carbapenem-resistant enterobacteria , Primer pairs of SEQ ID NOs: 9 and 10 for the IMP (-1, -3, -6, -10) gene specific for carbapenem-resistant enterobacteriaceae, IMP (-4, -26) gene specific for carbapenem-resistant enterobacteria Primer pairs of SEQ ID NOs: 11 and 12, and primer pairs of SEQ ID NOs: 13 and 14 for genes of IMP (-2, -8, -19, -20, -24) specific for carbapenem-resistant enterobacteriaceae Selected from the group At least one primer pair and a label of DNA polymerase, dNTPs, PCR buffer and PCR amplification products.

In a real-time PCR amplification kit for detecting carbapenem-resistant enterobacteria of one embodiment of the present invention, the labeling substance is a probe that specifically binds to the KPC gene specific for carbapenem-resistant enterobacteriaceae. A probe consisting of an oligonucleotide having or a nucleotide sequence complementary to such an oligonucleotide, a probe specifically binding to a VIM gene specific for carbapenem-resistant enterobacteria, an oligonucleotide having a nucleotide sequence of SEQ ID NO: 16 or such an oligonucleotide Probes consisting of oligonucleotides having complementary nucleotide sequences to nucleotides, brobes that specifically bind to NDM genes specific for carbapenem-resistant enterobacteria, oligonucleotides having the nucleotide sequence of SEQ ID NO: 17, or such oligonucleotides Probes consisting of oligonucleotides with complementary nucleotide sequences, probes that specifically bind to GES genes specific for carbapenem-resistant enterobacteria, oligonucleotides having the nucleotide sequence of SEQ ID NO: 18 or nucleotide sequences complementary to these oligonucleotides Probe consisting of an oligonucleotide having a probe that binds specifically to the IMP (-1, -3, -6, -10) gene specific for carbapenem-resistant enterobacteria, oligonucleotide having the nucleotide sequence of SEQ ID NO: 19 or such A probe consisting of an oligonucleotide having a nucleotide sequence complementary to an oligonucleotide, a probe specifically binding to an IMP (-4, -26) gene specific for carbapenem-resistant enterobacteria, an oligonucleotide having a nucleotide sequence of SEQ ID NO: 20, or Salts complementary to these oligonucleotides A probe consisting of an oligonucleotide having a sequence, and a probe specifically binding to a gene of IMP (-2, -8, -19, -20, -24) specific for carbapenem-resistant enterobacteriaceae; It may be labeled at one end, for example, 5 'end of at least one probe selected from the group consisting of an oligonucleotide having a probe or a probe consisting of an oligonucleotide having a base sequence complementary to such an oligonucleotide.

The real-time PCR amplification kit for detecting carbapenem-resistant enterobacteria of one embodiment of the present invention preferably comprises two probes labeled with a label that can be detected at different wavelengths. As such, when two probes labeled with a label that can be detected at different wavelengths are simultaneously included, two target genes specific to carbapenem-resistant enterobacteria may be detected at one time in one test tube.

As an example, one end of one probe, eg, 5 'end, is a label that can be detected at 470 nm, and one end of another probe, eg, 5' end, is a label that can be detected at 530 nm. It can be labeled with a substance. For example, the label that can be detected at 470nm may be FAM, and the label that can be detected at 530nm may be VIC. However, the present invention is not limited thereto, and a label having various detection wavelengths may be used.

In the present invention, the labeling substance is Cy5, Cy3, biotin-binding substance, EDANS (5- (2'-aminoethyl) amino-1-naphthalene sulfate), tetramethyltamine (TMR), tetramethyltamine isocyanate (TMRITC), x-rhodamine or Texas red, SYBR green I, VIC, FAM and the like. However, as described above, the present invention is not limited thereto, and various labeling materials such as various fluorescent materials, radioactive materials, or luminescent materials known in the art may be used.

According to the present invention, real-time PCR (real-time polymerase chain reaction) can be used to quickly and accurately examine whether carbapenem-resistant enterobacteriaceae is infected from a sample of a patient suspected of carbapenem-resistant enterobacteriaceae.

In addition, the present invention uses real-time PCR to detect high-specificity and sensitivity of carbapenem-resistant enterobacteria, which may be present in a sample of a suspected carbapenem-resistant enterobacteriaceae infection, on the basis of rapid, accurate and quantified data, and carbapenem resistance. Early detection of the presence of a small amount of carbapenem-resistant enterobacteriaceae in the early stages of intestinal bacterial infection enables proper dosing treatment at the early stage of infection, preventing the patient's treatment time or preventing the spread of infection in advance.

In addition, according to the present invention, the test result reading on the sample can be read as a value of the Ct value (or Cp value) by using the multiplex real-time PCR technology, so that the accurate result reading and the different wavelengths By using two probes labeled with a label that can be detected at the same time, it is possible to detect two target genes in one test tube at a time, thereby reducing the test time.

Therefore, the present invention has the advantage of being able to identify and manage carbapenem-resistant enterobacteriaceae early through a quick and accurate test for the infection of carbapenem-resistant enterobacteriaceae.

1 is a diagram showing the results of nucleotide sequence homology analysis of KPC genotype.
Figure 2 shows the results of nucleotide sequence homology analysis of the VIM genotype.
3 shows sequence information of the NDM genotype NDM-1.
4 shows the results of nucleotide sequence homology analysis of the GES genotype.
5 shows the results of nucleotide sequence homology analysis of the IMP genotype.
Figure 6 is a photograph of the results of electrophoresis on agarose gels of amplification products for the cloning of CRE (cabapenem resistant enterobacteriaceae) genes.
7 is a template of plasmids transformed with NDM, KPC, IMP, GES and VIM target genes respectively, and after amplification of each target gene using respective CRE detection primers, the amplification products are transferred to agarose gel. It is a photograph of the result.
8 is a method for testing carbapenem-resistant enterobacteriaceae of an embodiment of the present invention, using a KPC gene transforming plasmid and a VIM gene transforming plasmid as a template, and using a detection primer and a probe for each of the KPC gene and the VIM gene, respectively. This is an amplification graph obtained by performing a flex real-time PCR.
9 is a method for testing carbapenem-resistant enterobacteriaceae of an embodiment of the present invention, using a NDM transgenic plasmid and a GES transgenic plasmid as a template, and using a detection primer and a probe for each of the NDM and GES genes, respectively. This is an amplification graph obtained by performing a flex real-time PCR.
10 is a method for testing carbapenem-resistant enterobacteriaceae of an embodiment of the present invention, using an IMP-6, -4, -2 gene transforming plasmid as a template and detecting primers for an IMP-6, -4, -2 gene Amplification curve graph obtained by performing multiplex real-time PCR using and probe.
FIG. 11 is an amplification curve graph obtained by performing multiplex real-time PCR on a carbapenem-resistant enterobacteriaceae test method according to an embodiment of the present invention.
12 is an amplification curve graph obtained by performing multiplex real-time PCR on a carbapenem-resistant enterobacteriaceae test method according to an embodiment of the present invention.
Figure 13 is a graph showing the detection limit copy number analysis results according to the carbapenem resistance enterobacteriaceae test method using multiplex real-time PCR of the present invention. As shown in FIG. 13, unlike the disk diffusion method, the present invention can significantly detect a small copy number of carbapenem-resistant enterobacteriaceae gene transforming plasmid, which enables detection of carbapenem-resistant enterobacteria with high sensitivity as well as the degree of infection. It can be seen that (infection risk index) can be quantified.

Hereinafter, the present invention will be described in more detail based on the embodiments of the present invention. It should be understood that the following embodiments of the present invention are only for embodying the present invention and do not limit or limit the scope of the present invention. It will be understood by those of ordinary skill in the art that various changes in form and details may be made therein without departing from the spirit and scope of the invention as defined by the appended claims. The references cited in the present invention are incorporated herein by reference.

Example

Example 1: Securing sequence information of the CRE gene

The base sequences for NDM, KPC, IMP, GES, and VIM gene analysis possessed by carbapenem-resistant enterobacteria were obtained by genotype of each gene registered in NCBI, and the common sequences were analyzed (BioEdit, USA). ) Was analyzed using (Fig. 1 to Fig. 5). Based on the analyzed sequences, the Primer Express (AppliedBiosystems, USA) program was used to design primers that can detect multiple genotypes of each gene and fluorescently labeled probes that can identify reaction products.

Design primers and probes from the common sequences of the KPC-1, -2, -3, -4, -5, -6, -8, -9, -11 genotypes to determine KPC-1, -2, -3, -4, -5, -6, -8, -9, -11 genotypes were detected at one time using one primer set (SEQ ID NO: 1 and SEQ ID NO: 2) and one probe (SEQ ID NO: 15) . Also, VIM-1, -2, -3, -4, -5, -6, -7, -8, -9, -10, -11, -12, -14, -15, -16, -18 By designing primers and probes in common sequences of the -19, -20, -23, -24, -26 genotypes, VIM-1, -2, -3, -3, -4, -5, -6, -7, -8, -9, -10, -11, -12, -14, -15, -16, -18, -19, -20, -23, -24, -26 genotype 1 primer set (SEQ ID NO: 3 and SEQ ID NO: 4) and one probe (SEQ ID NO: 16) were used for detection at one time.

However, NDM has only one genotype of NDM-1 detected since there are not enough reports of other genotypes (primerset: SEQ ID NO: 5 and SEQ ID NO: 6; probe: SEQ ID NO: 17), GES-1, -2 , -3, -4, -5, -7, -12, -13, -15, -16 genotype using one primer set (SEQ ID NO: 7 and SEQ ID NO: 8) and one probe (SEQ ID NO: 18) To be detected at a time.

In addition, in the case of the IMP gene, unlike the previous four genes, the genome sequence of each genotype was analyzed to be large, and thus, three primer sets (SEQ ID NO: 9 and SEQ ID NO: 10, SEQ ID NO: 11 and SEQ ID NO: 12, SEQ ID NO: 13) were analyzed. And IMP-1, -2, -3, -4, -6, -8, -10, -19 using SEQ ID NO: 14) and three probes (SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21). The, -20, -24, -26 genotypes were detected (see Table 1 and Table 2 below).

Table 1: Primer Sequences for CRE Gene Detection

Table 2: Probe Sequence for CRE Gene Detection

Example 2: Clone of CRE Gene

In order to secure the CRE gene, genomic DNA of a strain verified to be positive for CRE infection was collected at a university hospital, and specific primers (SEQ ID NOs: 22 to 33) of each gene were used (see Table 3 below). KPC, IMP, GES and VIM genes were amplified.

Table 3: Primer Sequences for Cloning the CRE Gene

PCR amplification was repeated 10 times at 94 ° C. for 15 seconds at 94 ° C. and 40 times at 60 ° C. for 1 minute. Each amplified product amplified by using a 1% agarose gel was confirmed whether the non-specific band formation (see Figure 6). PCR amplification products of the identified CRE gene was purified using GeneAll Expin Gel SV (GeneAll, Korea) kit, and the purified amplification products were inserted into pGEM-T easy vector (Promega, USA). E. coli was transformed.

Prepared transformed E. coli was incubated for 16 hours in LB medium and purified for plasmid DNA using GeneAll plasmid prep kit (School Genetech, Korea). The obtained sequence information was analyzed by blast search, and as a result, it was confirmed that the common base sequences of various genotypes of NDM, KPC, IMP, GES, and VIM genes were included (SEQ ID NOs 37 to SEQ ID NOs: 42). In the case of IMP-2 gene which contains the common nucleotide sequence of IMP-2, -8, -19, -20, -24 genotype, the nucleotide sequence was analyzed because it could not receive the genomic DNA of the tested bacteria in Korea. On the basis of this, artificially requested gene synthesis service (Bioneer, Korea) to obtain a transgenic clone (SEQ ID NO: 43).

Example 3: Identification of CRE Detection Primers

In order to confirm whether the CRE detection primers (SEQ ID NO: 1 to SEQ ID NO: 14) designed in Example 1 specifically respond to the gene, the NDM, KPC, IMP, GES, and VIM genes prepared in Example 2 were transformed. The plasmid DNA was used as a template, and PCR amplification was carried out using primers for detecting CRE of the genes. The amplification products were confirmed by electrophoresis using agarose gel (3%). PCR amplification was repeated 10 times at 94 ° C. for 15 seconds at 94 ° C. and 40 times at 62 ° C. for 1 minute.

As a result of confirming the PCR amplification product was not able to confirm the formation of the non-specific band, the size of the normal amplification product was confirmed (Fig. 7).

Example 4 Identification of KPC and VIM Gene Detection Primers and Probes Using Multiplex Real Time PCR

Real-time PCR is a method of applying fluorescent material to PCR, and amplifies the target genes present in the sample during the reaction and detects the emission level of the fluorescent material in real time and quantitatively analyzes it. This is a method to analyze the presence or absence of amplification of the target gene and its pattern quickly and accurately. Real-time PCR methods are largely divided into a method using a TaqMan probe and a method using SYBR Green I. In embodiments of the present invention, real-time PCR was performed using a TaqMan probe, but the present invention is not limited thereto.

The basic principle of real-time PCR is the detection and quantification of fluorescence which is carried out in real time every cycle of PCR by the principle of polymerase and Fluorescence Resonance Energy Transfer (FRET). Quantification in real-time PCR is a method of measuring amplification products in real time within the exponential phase range by monitoring the progress of each cycle during PCR. An important concept is Ct (threshold cycle) or Cp (crossing). point). This value is the number of cycles at which the amount of fluorescence produced by the separation of the reader increases noticeably beyond the baseline level, which is the initial template amount (NDM, KPC, IMP, GES, the number of copies of the VIM gene).

For example, real-time PCR can be used to measure the change in fluorescence intensity of a label over the entire PCR amplification period of an internal control that is amplified regardless of whether the sample is positive or negative. Recovery (Cp value or Ct value) can be analyzed and a standard curve of fluorescence intensity can be obtained. The standard curves of Cp or Ct and fluorescence intensity of the internal control are clearly distinguished from the curve patterns of Cp or Ct and fluorescence intensity of the negative control to provide a criterion for determining whether the unknown sample is positive or negative. Can be. Therefore, PCR amplification of samples containing NDM, KPC, IMP, GES and / or VIM genes to measure the change in fluorescence intensity of the label for the entire PCR amplification period and the number of PCR reactions (Cp value) to reach a constant fluorescence intensity Or Ct value) and obtain a standard curve of fluorescence intensity, and then compare Np, KPC, IMP, GES and / or VIM in the sample by comparing the Cp value or Ct value of the internal control and negative control with the curve pattern of fluorescence intensity. The presence or absence of the gene, i.e., whether the subject is infected with carbapenem-resistant enterobacteriaceae, can be quickly and accurately tested for high specificity and sensitivity based on accurate and quantified data.

Specifically, the KPC primer pair (SEQ ID NO: 1 and SEQ ID NO: 2), VIM primer pair (SEQ ID NO: 3 and SEQ ID NO: 3) identified in Example 3 to simultaneously detect the KPC gene and VIM gene in one test tube (Tube) 4) KPC probes (SEQ ID NO: 15) and VIM probes (SEQ ID NO: 16) labeled with different fluorescent materials (different emission wavelengths) were used, and real time PCR was performed using real time PCR equipment (LG Life Sciences, Korea) using the transformed plasmid DNA prepared in Example 2 as a template.

As a PCR amplification composition, a commercially available HS Prime Taq premix (Gennet Bio, Korea) was used, and the amplification reaction was prepared by adding the aforementioned KPC primer / probe and VIM primer / probe. The amplification process was repeated for 10 minutes at 94 ℃, 15 seconds at 94 ℃, was repeated 35 times for 1 minute at 60 ℃. In the reading of real-time PCR amplification reactions, a fluorescent material fluorescing at different wavelengths is labeled at the 5 'end of each probe to detect internal control genes, KPC genes and VIM genes. Three real-time PCR amplification reactions were read out, and MGB (Minor Groove Binder) was coupled to the 3 'end of each probe to improve the binding force between the probe and the template.

In the identification of three real-time PCR amplification reactions generated in one test tube, the KPC gene can be identified by the fluorescence value at 470 nm wavelength and the VIM gene can be identified by the fluorescence value at 530 nm wavelength. In addition, the internal control (NC_003071: CPN60A (chloroplast / 60 kDa chaperonin alpha Subunit)) was confirmed by the fluorescence value coming from the 630 nm wavelength. In addition, by using the internal control as described above, it was possible to verify the effectiveness of the amplification reaction regardless of the positive and negative results of the unknown sample. In the embodiments of the present invention (including the following examples), internal control is amplified by the following primer pair (SEQ ID NO: 34 and SEQ ID NO: 35) as an example, and the 5 'end is labeled with Cy5 as follows. Fluorescent labeling was carried out by a probe (SEQ ID NO: 36) to measure the fluorescence value.

IC F-primer: 5'-GGG ACA ACC ACT GCG TCT AT-3 '(SEQ ID NO: 34)

IC R-primer: 5'-CAC AAG GAA GGT CAC TGC AA-3 '(SEQ ID NO: 35)

IC probe: Cy5- 5'-CGT CAC TTC TGG TGC GAA TCC CGT-3 '(SEQ ID NO: 36)

Real-time PCR reactions were able to clarify the classification of the three reactants in one tube, it was confirmed that the false positive or false negative results of the three reactants (Fig. 8).

Example 5 Identification of NDM and GES Gene Detection Primers and Probes Using Multiplex Real Time PCR

NDM primer pair (SEQ ID NO: 5 and SEQ ID NO: 6), GES primer pair (SEQ ID NO: 7 and SEQ ID NO: 8) identified in Example 3 to simultaneously detect NDM gene and GES gene in one test tube, and NDM probes (SEQ ID NO: 17) and GES probes (SEQ ID NO: 18) labeled with different fluorophores (different emission wavelengths) were used, and real-time PCR was performed using real-time PCR equipment (LG Life Science, Korea). Using the transformed plasmid DNA prepared in Example 2 as a template.

Composition and amplification process of the amplification reaction was carried out in the same manner as in Example 4.

In the reading of real-time PCR amplification reactions, a fluorescent material fluorescing at different wavelengths is labeled at the 5 'end of each of the probes for detecting the internal control gene, the NDM gene and the GES gene. Three real-time PCR amplification reactions were read out, and MGB (Minor Groove Binder) was coupled to the 3 'end of each probe to improve the binding force between the probe and the template.

In the identification of three real-time PCR amplification reactions generated in one test tube, the fluorescence value from the 470 nm wavelength for the NDM gene and the fluorescence value from the 530 nm wavelength for the GES gene can be confirmed. In addition, the internal control was able to identify the fluorescence value coming from the 630 nm wavelength. In addition, by using the internal control as described above, it was possible to verify the effectiveness of the amplification reaction regardless of the positive and negative results of the unknown sample.

Real-time PCR reactions were able to clarify the classification of the three reactants in one tube, it was confirmed that the false positive or false negative results of the three reactants (Fig. 9).

Example 6: Identification of IMP Gene Detection Primers and Probes by Multiplex Real Time PCR

IMP-6 primer pair (SEQ ID NO: 9 and SEQ ID NO: 10), IMP-4 primer pair (SEQ ID NO: 11 and SEQ ID NO: 12) identified in Example 3 to detect the IMP gene in one test tube (Tube), IMP-6 probe (SEQ ID NO: 19), IMP-4 probe (SEQ ID NO: 20), labeled with an IMP-2 primer pair (SEQ ID NO: 13 and SEQ ID NO: 14) and different fluorescents (with different emission wavelengths); IMP-2 probe (SEQ ID NO: 21) was used, and real-time PCR was performed using the transformed plasmid DNA prepared in Example 2 as a template using a real-time PCR equipment (LG Life Science, Korea).

Composition and amplification process of the amplification reaction was carried out in the same manner as in Example 4.

In reading real-time PCR amplification reactions, the internal control gene, IMP-6 gene (used to encompass IMP-1, -3, -6, -10 genotypes), IMP-2 gene ( Probes to detect the IMP-2, -8, -19, -20, -24 genotypes) and the IMP-4 gene (used to encompass the IMP-4, -26 genotypes) 5 'end of the nucleotide sequence of the labeling fluorescent material fluorescing at different wavelengths (but IMP-6 gene and IMP-4 gene are all labeled with a fluorescent material, for example, FAM fluorescence at the same wavelength) In addition, three real-time PCR amplification reactions were read, and MGB (Minor Groove Binder) was coupled to each probe 3 'end to improve the binding force between the probe and the template.

In the identification of three real-time PCR amplification reactions generated in one test tube, it is possible to confirm the fluorescence value of 470 nm wavelength for IMP-6, -4 gene and 530 nm wavelength for IMP-2 gene. The fluorescence value from the fluorescence value can be identified by the internal control, and the fluorescence value from the 630 nm wavelength can be confirmed. In addition, by using the internal control as described above, it was possible to verify the effectiveness of the amplification reaction regardless of the positive and negative results of the unknown sample.

Real-time PCR reactions were able to clarify the classification of the three reactants in one tube, it was confirmed that the false positive or false negative results of the three reactants (Fig. 10).

Example 7: Confirmation of CRE Detection in Specimen Using Multiplex Real-Time PCR

Using the CRE detection primer pairs / probes verified in Example 4, Example 5 and Example 6, it was confirmed whether the CRE related genes were detectable in the specimen using multiplex real time PCR. The samples used were cultured on agar medium using clinical samples obtained from patients suspected of CRE infection in university hospitals, and then extracted from the genome DNA of the bacteria. The genotypes of the NDM, KPC, IMP, GES and VIM genes among the CRE genes were verified by the test and sequencing methods.

Seventeen CRE positive specimens and six CRE negative specimens were used.

CRE positive samples were two KPC-2 genotypes, two KPC-3 genotypes, three GES-5 genotypes, four NDM-1 genotypes, two VIM-2 genotypes, and one VIM-3 genotype. Samples, one IMP-4 genotype sample and two IMP-6 genotype samples were used. NDM-1, KPC, IMP, GES and VIM genes were identified using three test tubes for the identification of one specimen.

Tube 1 (Tube 1) shown in Table 4 and Table 5 was confirmed KPC gene and VIM gene, Tube 2 (Tube 2) was confirmed NDM gene and GES gene, Tube 3 (Tube 3) IMP gene Confirmed.

In addition, the reaction was validated using Internal Control (IC) for both Tube 1, Tube 2 and Tube 3. The reaction composition was prepared by adding HS Prime Taq premix (Gennet Bio, Korea) and the corresponding primer pairs / probes of Tube 1, Tube 2 and Tube 3.

The reaction process was repeated for 10 minutes at 94 ℃, 15 seconds at 94 ℃, was repeated 35 times 1 minute at 60 ℃, it was performed using a real-time PCR equipment (LG Life Science, Korea). The real-time PCR reaction product was read in a positive / negative manner by the Ct value of each reactant and the amplification curve of the reactants obtained when the limit value of 0.05 was given (Table 4, Table 5, FIGS. 11 and 12). Reference). The serial numbers following IMP in Table 4 and Table 5 are for distinguishing genotypes of IMP genes, IMP13461026 represents genotypes of IMP-1, -3, -4, -6, -10, -26, and IMP28192024 represents IMP. Genotype of -2, -8, -19, -20-24. In tube 3, IMP-1, -3, -4, -6, -10, -26 genotypes are labeled with FAM and can be identified by fluorescence at 470 nm wavelength, IMP-2, -8, -19,- Genotypes of 20, -24 are labeled with VIC and can be identified by their fluorescence values at 530 nm wavelength.

Table 4: Confirmation of CRE negative sample results using multiplex real-time PCR

Table 5: Confirmation of CRE positive specimens using multiplex real-time PCR

In summary, the results of the 23 samples were confirmed to be identical using multiplex real-time PCR. Therefore, multiplex real-time PCR can be used to detect various genotypes of NDM, KPC, IMP, GES and VIM genes of CRE more quickly and accurately.

Example 8: Confirmation of detection limit copy number (detection sensitivity) using the multiplex real-time PCR method of the present invention

Detection limit copy number was confirmed by multiplex PCR using the plasmid DNA of each CRE gene prepared in Example 2. The plasmid DNA cloned with NDM, IMP, VIM, KPC and GES target genes was diluted stepwise with sterile distilled water to prepare each sample (positive standard) from 5 × 10 ⁶ copies to 5 × 6.25 copies. Detection limit test of the multiplex real-time PCR method of the present invention was performed. The multiplex real-time PCR method of the present invention is carried out by repeating the amplification reaction using a real-time PCR equipment (LG Life Science, Korea) for 10 minutes at 94 ℃, 15 seconds at 94 ℃, 35 times at 60 ℃ 1 minute It was.

Reading of the amplification reaction resulted in a positive / negative reading with an amplification curve of each reaction obtained when the limit value was 0.05 (FIG. 13). The test results confirmed that KPC can detect 125 copies, VIM 63 copies, and NDM and GES up to 250 copies. IMP (1,3,4,6,10,26) is 63 copies, and IMP (2,8) , 19,20,24) confirmed that up to 32 copies were detectable. Therefore, as shown in Figure 13, it was confirmed the effectiveness and high sensitivity of the multiplex real-time PCR method of the present invention.

Taken together, the present invention, unlike the disk diffusion method, can significantly detect a small copy number of carbapenem-resistant enterobacteriaceae gene transforming plasmids, which can detect carbapenem-resistant enterobacteria with high sensitivity as well as the degree of infection (infection). It is also possible to quantify the risk index. Therefore, the present invention uses real-time PCR to detect high-specificity and sensitivity of carbapenem-resistant enterobacteria, which may be present in a sample of suspected carbapenem-resistant enterobacteriaceae infection, based on rapid, accurate and quantified data, and carbapenem resistance. Early detection of the presence of a small amount of carbapenem-resistant enterobacteriaceae in the early stages of intestinal bacterial infection enables the appropriate dosing treatment at the beginning of the infection, thereby preventing the patient's treatment time or preventing the spread of infection in advance.

Although the present invention has been described with reference to the above embodiments, the present invention is not limited thereto. It will be understood by those skilled in the art that modifications and variations may be made without departing from the spirit and scope of the invention, and that such modifications and variations are also contemplated by the present invention.

<110> GENOCHECK CO., LTD. <120> Method and kit for detecting carbapenem resistant          enterobacteriaceae using real-time PCR <130> P11-0252KR <150> KR 10-2011-0066620 <151> 2011-07-05 <160> 43 <170> Kopatentin 1.71 <210> 1 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> KPC F primer <400> 1 gcaatgtgct caacgttcaa g 21 <210> 2 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> KPC R primer <400> 2 gtgcctgagt caattctttc aaag 24 <210> 3 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> VIM F primer <400> 3 aatggtctca ttgtccgtga tg 22 <210> 4 <211> 17 <212> DNA <213> Artificial Sequence <220> <223> VIM R primer <400> 4 tcgcacccca cgctgta 17 <210> 5 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> NDM F primer <400> 5 tgcatgcccg gtgaaatc 18 <210> 6 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> NDM R primer <400> 6 gtcgccagtt tccatttgct 20 <210> 7 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> GES F primer <400> 7 gcaatgtgct caacgttcaa g 21 <210> 8 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> GES R primer <400> 8 gtgcctgagt caattctttc aaag 24 <210> 9 <211> 21 <212> DNA <213> Artificial Sequence <220> IMP F-6 primer <400> 9 cgatctatcc ccacgtatgc a 21 <210> 10 <211> 23 <212> DNA <213> Artificial Sequence <220> IMP R-6 primer <400> 10 ggcttgaacc ttaccgtctt ttt 23 <210> 11 <211> 20 <212> DNA <213> Artificial Sequence <220> IMP F-4 primer <400> 11 cacttggttt gtggaacgtg 20 <210> 12 <211> 22 <212> DNA <213> Artificial Sequence <220> IMP R-4 primer <400> 12 gggatggatt gagaattaag cc 22 <210> 13 <211> 16 <212> DNA <213> Artificial Sequence <220> <223> IMP F-2 primer <400> 13 ggagcgcggc tataaa 16 <210> 14 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> IMP R-2 primer <400> 14 tgctgtcgct atggaaatgt g 21 <210> 15 <211> 14 <212> DNA <213> Artificial Sequence <220> <223> KPC probe <400> 15 ccgccaattt gttg 14 <210> 16 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> VIM probe <400> 16 tgatgagttg cttttgattg 20 <210> 17 <211> 13 <212> DNA <213> Artificial Sequence <220> <223> NDM probe <400> 17 cccgacgatt ggc 13 <210> 18 <211> 15 <212> DNA <213> Artificial Sequence <220> <223> GES probe <400> 18 ttccgctagc cgcgc 15 <210> 19 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> IMP probe-6 <400> 19 ctgaattaac aaatgaactg c 21 <210> 20 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> IMP probe-4 <400> 20 agtatttcct ctcattttc 19 <210> 21 <211> 17 <212> DNA <213> Artificial Sequence <220> <223> IMP probe-2 <400> 21 tcaaaggcac tatttcc 17 <210> 22 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> KPC cloF primer <400> 22 cagctcattc aagggctttc 20 <210> 23 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> KPC cloR primer <400> 23 tagtcatttg ccgtgccata 20 <210> 24 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> VIM cloF primer <400> 24 aggtccggct ttaccagatt 20 <210> 25 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> VIM cloR primer <400> 25 ccatagagca cactcgcaga 20 <210> 26 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> NDM cloF primer <400> 26 catttgcggg gtttttaatg 20 <210> 27 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> NDM cloR primer <400> 27 tagtgctcag tgtcggcatc 20 <210> 28 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> GES cloF primer <400> 28 gagaagctag agcgcgaaaa 20 <210> 29 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> GES cloR primer <400> 29 tatctctgag gtcgccaggt 20 <210> 30 <211> 20 <212> DNA <213> Artificial Sequence <220> IMP cloF-6 primer <400> 30 aaaggcagca tttcctctca 20 <210> 31 <211> 20 <212> DNA <213> Artificial Sequence <220> IMP cloR-6 primer <400> 31 caagagtgat gcgtctccaa 20 <210> 32 <211> 20 <212> DNA <213> Artificial Sequence <220> IMP cloF-4 primer <400> 32 ggcgttgttc ctaaacatgg 20 <210> 33 <211> 20 <212> DNA <213> Artificial Sequence <220> IMP cloR-4 primer <400> 33 gatgcgtctc cagcttcact 20 <210> 34 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Internal Control F-primer <400> 34 gggacaacca ctgcgtctat 20 <210> 35 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Internal Control R-primer <400> 35 cacaaggaag gtcactgcaa 20 <210> 36 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Internal Control probe <400> 36 cgtcacttct ggtgcgaatc ccgt 24 <210> 37 <211> 534 <212> DNA <213> Artificial Sequence <220> <223> KPC gene clone <400> 37 cagctcattc aagggctttc ttgctgccgc tgtgctggct cgcagccagc agcaggccgg 60 cttgctggac acacccatcc gttacggcaa aaatgcgctg gttccgtggt cacccatctc 120 ggaaaaatat ctgacaacag gcatgacggt ggcggagctg tccgcggccg ccgtgcaata 180 cagtgataac gccgccgcca atttgttgct gaaggagttg ggcggcccgg ccgggctgac 240 ggccttcatg cgctctatcg gcgataccac gttccgtctg gaccgctggg agctggagct 300 gaactccgcc atcccaggcg atgcgcgcga tacctcatcg ccgcgcgccg tgacggaaag 360 cttacaaaaa ctgacactgg gctctgcact ggctgcgccg cagcggcagc agtttgttga 420 ttggctaaag ggaaacacga ccggcaacca ccgcatccgc gcggcggtgc cggcagactg 480 ggcagtcgga gacaaaaccg gaacctgcgg agtgtatggc acggcaaatg acta 534 <210> 38 <211> 460 <212> DNA <213> Artificial Sequence <220> <223> VIM gene clone <400> 38 aggtccggct ttaccagatt gccgatggtg tttggtcgca tatcgcaacg cagtcgtttg 60 atggcgcagt ctacccgtcc aatggtctca ttgtccgtga tggtgatgag ttgcttttga 120 ttgatacagc gtggggtgcg aaaaacacag cggcacttct cgcggagatt gagaagcaaa 180 ttggacttcc tgtaacgcgt gcagtctcca cgcactttca tgacgaccgc gtcggcggcg 240 ttgatgtcct tcgggcggct ggggtggcaa cgtacgcatc accgtcgaca cgccggctag 300 ccgaggtaga ggggaacgag attcccacgc actctctaga aggactctca tcgagcgggg 360 acgcagtgcg cttcggtcca gtagaactct tctatcctgg tgctgcgcat tcgaccgaca 420 acttagttgt gtacgtcccg tctgcgagtg tgctctatgg 460 <210> 39 <211> 729 <212> DNA <213> Artificial Sequence <220> <223> NDM gene clone <400> 39 tcgcatttgc ggggttttta atgctgaata aaaggaaaac ttgatggaat tgcccaatat 60 tatgcacccg gtcgcgaagc tgagcaccgc attagccgct gcattgatgc tgagcgggtg 120 catgcccggt gaaatccgcc cgacgattgg ccagcaaatg gaaactggcg accaacggtt 180 tggcgatctg gttttccgcc agctcgcacc gaatgtctgg cagcacactt cctatctcga 240 catgccgggt ttcggggcag tcgcttccaa cggtttgatc gtcagggatg gcggccgcgt 300 gctggtggtc gataccgcct ggaccgatga ccagaccgcc cagatcctca actggatcaa 360 gcaggagatc aacctgccgg tcgcgctggc ggtggtgact cacgcgcatc aggacaagat 420 gggcggtatg gacgcgctgc atgcggcggg gattgcgact tatgccaatg cgttgtcgaa 480 ccagcttgcc ccgcaagagg ggatggttgc ggcgcaacac agcctgactt tcgccgccaa 540 tggctgggtc gaaccagcaa ccgcgcccaa ctttggcccg ctcaaggtat tttaccccgg 600 ccccggccac accagtgaca atatcaccgt tgggatcgac ggcaccgaca tcgcttttgg 660 tggctgcctg atcaaggaca gcaaggccaa gtcgctcggc aatctcggtg atgccgacac 720 tgagcacta 729 <210> 40 <211> 439 <212> DNA <213> Artificial Sequence <220> <223> GES gene clone <400> 40 gagaagctag agcgcgaaaa agcagctcag atcggtgttg cgatcgtcga tccccaagga 60 gagatcgtcg cgggccaccg aatggcgcag cgttttgcaa tgtgctcaac gttcaagttt 120 ccgctagccg cgctggtctt tgaaagaatt gactcaggca ccgagcgggg ggatcgaaaa 180 ctttcatatg ggccggacat gatcgtcgaa tggtctcctg ccacggagcg gtttctagca 240 tcgggacaca tgacggttct cgaggcagcg caagctgcgg tgcagcttag cgacaatggg 300 gctactaacc tcttactgag agaaattggc ggacctgctg caatgacgca gtattttcgt 360 aaaattggcg actctgtgag tcggctagac cggaaagagc cggagatgag cgacaacaca 420 cctggcgacc tcagagata 439 <210> 41 <211> 408 <212> DNA <213> Artificial Sequence <220> <223> IMP-6 gene clone <400> 41 aaaggcagca tttcctctca ttttcatagc gacagcacgg gcggaataga gtggcttaat 60 tctcgatcta tccccacgta tgcatctgaa ttaacaaatg aactgcttaa aaaagacggt 120 aaggttcaag ccacaaattc atttagcgga gttaactatt ggctagttaa aaataaaatt 180 gaagtttttt atccaggccc gggacacact ccagataacg tagtggtttg gttgcctgaa 240 aggaaaatat tattcggtgg ttgttttatt aaaccgtacg gtttaggcaa tttgggtgac 300 gcaaatatag aagcttggcc aaagtccgcc aaattattaa agtccaaata tggtaaggca 360 aaactggttg ttccaggtca cagtgaagtt ggagacgcat cactcttg 408 <210> 42 <211> 527 <212> DNA <213> Artificial Sequence <220> <223> IMP-4 gene clone <400> 42 ggcgttgttc ctaaacatgg tttggttgtt cttgtagatg ctgaagctta tctaattgac 60 actccattta cggctaaaga tactgaaaag ttagtcactt ggtttgtgga acgtggctat 120 aaaataaaag gcagtatttc ctctcatttt catagtgaca gcacgggcgg aatagagtgg 180 cttaattctc aatccatccc cacgtatgcg tctgaattaa ctaatgagct gcttaaaaaa 240 gacggtaagg ttcaagctaa aaattcattt ggcggggtta actattggct agttaaaaat 300 aaaattgaag ttttttatcc aggcccagga cacactccag ataacctagt agtttggctg 360 cctgaaagga aaatattatt cggtggttgt tttattaaac cgtacggtct aggtaatttg 420 ggtgacgcaa atttagaagc ttggccaaag tccgctaaat tattaatatc caaatatggt 480 aaggcaaaac tggttgttcc aagtcacagt gaagctggag acgcatc 527 <210> 43 <211> 701 <212> DNA <213> Artificial Sequence <220> <223> IMP-2 gene clone <400> 43 ttatttgttt tatgtgtatg cttcctttgt agcattactg ccgcgggagc gcgtttgcct 60 gatttaaaaa tcgagaagct tgaagaaggt gtttatgttc atacatcgtt cgaagaagtt 120 aacggttggg gtgttgtttc taaacacggt ttggtggttc ttgtaaacac tgacgcctat 180 ctgattgaca ctccatttac tgctacagat actgaaaagt tagtcaattg gtttgtggag 240 cgcggctata aaatcaaagg cactatttcc tcacatttcc atagcgacag cacaggggga 300 atagagtggc ttaattctca atctattccc acgtatgcat ctgaattaac aaatgaactt 360 cttaaaaaag acggtaaggt gcaagctaaa aactcattta gcggagttag ttattggcta 420 gttaaaaata aaattgaagt tttttatccc ggcccggggc acactcaaga taacgtagtg 480 gtttggttac ctgaaaagaa aattttattc ggtggttgtt ttgttaaacc ggacggtctt 540 ggtaatttgg gtgacgcaaa tttagaagct tggccaaagt ccgccaaaat attaatgtct 600 aaatatgtta aagcaaaact ggttgtttca agtcatagtg aaattgggga cgcatcactc 660 ttgaaacgta catgggaaca ggctgttaaa gggctaaatg a 701

Claims (9)

Preparing a genetic sample from the sample,
Primer pairs of SEQ ID NOS: 1 and 2 for the KPC gene specific for carbapenem-resistant enterobacteria, primer pairs of SEQ ID NOs: 3 and 4 for the VIM gene specific for carbapenem-resistant enterobacteria, using the prepared gene sample as a template, Primer pairs of SEQ ID NOs: 5 and 6 for NDM genes specific for carbapenem-resistant enterobacteria, primer pairs of SEQ ID NOs: 7 and 8 for GES genes specific for carbapenem-resistant enteric bacteria, and IMP (- Primer pairs of SEQ ID NOs: 9 and 10 for 1, -3, -6, -10) genes, primer pairs of SEQ ID NOs: 11 and 12 for IMP (-4, -26) genes specific for carbapenem-resistant enterobacteriaceae, And amplifying at least one primer pair selected from the group consisting of primer pairs of SEQ ID NOs: 13 and 14 for genes of IMP (-2, -8, -19, -20, -24) specific for carbapenem-resistant enterobacteriaceae; Be And cover penem step of tolerance in relation to the specific genes to jangnaegyun using a marker that indicates the gene jeungpokryang, amplified to cover penem-resistant PCR in real time (Real-time) the specific gene to jangnaegyun in the gene sample in the sample,
After amplifying a gene specific for the carbapenem-resistant enterobacteriaceae, measuring the degree of infection of the carbapenem-resistant enterococci in the sample based on the amount of gene amplification specific to the carbapenem-resistant enterobacteria using the labeling substance;
Cabbapenem-resistant enterobacteriaceae using a real-time PCR, comprising the step of determining whether the infection in the sample carbapenem-resistant enteric bacteria based on the measured degree of carbapenem-resistant enterobacteriaceae. The method of claim 1,
The labeling substance is a fluorescent substance, the carbapenem resistance enterobacterial test method using a real-time PCR, characterized in that by measuring the intensity of fluorescence from the labeling material to calculate the amplification amount of the gene specific for carbapenem-resistant enterobacteria. The method of claim 2,
Before amplification, a positive standard, which is a gene sample that knows the copy number of a gene specific for carbapenem-resistant enterobacteria, is a primer pair corresponding to a gene specific for the carbapenem-resistant enterobacteria, and a gene specific for the amplified carbapenem-resistant enterobacteria. Amplifying by real-time PCR using a labeling material representing the amount of gene amplification in relation to
Measuring a change in fluorescence intensity of the labeling substance over the entire PCR amplification period of the positive standard, and analyzing the number of PCR reactions (Cp value or Ct value) attaining a constant fluorescence intensity;
Matching the copy number of the positive standard with a Cp value or a Ct value from a standard curve obtained by measuring a change in fluorescence intensity of the labeling substance;
The change in fluorescence intensity of the labeling substance over the entire PCR amplification period of the gene specific for the carbapenem-resistant enterobacteriaceae present in the sample of the sample was measured, and the number of PCR reactions (Cp value or Ct that reached a constant fluorescence intensity) was measured. Value),
The degree of infection of carbapenem-resistant enterococci present in the sample by mapping the Cp or Ct values obtained by PCR amplifying the gene specific for the carbapenem-resistant enterobacteria present in the sample of the sample to the copy number of the positive standard. Carbapenem resistance enterobacterial test method using real-time PCR further comprising the step of quantitatively measuring. 4. The method according to any one of claims 1 to 3,
The labeling substance is a probe that specifically binds to the KPC gene specific for carbapenem-resistant enterobacteria, an oligonucleotide having a nucleotide sequence of SEQ ID NO: 15 or a probe consisting of an oligonucleotide having a nucleotide sequence complementary to such oligonucleotide, carbapenem A probe specifically binding to a VIM gene specific for resistant enterobacteria, comprising a oligonucleotide having a nucleotide sequence of SEQ ID NO: 16 or an oligonucleotide having a nucleotide sequence complementary to such an oligonucleotide, a NDM specific for carbapenem-resistant enterobacteria A probe specifically binding to a gene, the probe consisting of an oligonucleotide having a nucleotide sequence of SEQ ID NO: 17 or an oligonucleotide having a nucleotide sequence complementary to such an oligonucleotide, carbapenem-resistant intestine A probe that specifically binds to a GES gene specific to a bacterium and comprises an oligonucleotide having a nucleotide sequence of SEQ ID NO: 18 or an oligonucleotide having a nucleotide sequence complementary to such an oligonucleotide, and an IMP specific to carbapenem-resistant enterobacteria ( -1, -3, -6, -10) A probe specifically binding to a gene, a probe consisting of an oligonucleotide having a nucleotide sequence of SEQ ID NO: 19 or an oligonucleotide having a nucleotide sequence complementary to such oligonucleotide, carbapenem A probe specifically binding to an IMP (-4, -26) gene specific for resistant enterobacteria, comprising a oligonucleotide having a nucleotide sequence of SEQ ID NO: 20 or an oligonucleotide having a nucleotide sequence complementary to such oligonucleotide, and Specific to Carbapenem-resistant Enterobacteriaceae Oligonucleotide having a nucleotide sequence of SEQ ID NO: 21 or an oligonucleotide having a nucleotide sequence complementary to such an oligonucleotide as a probe that specifically binds to a gene of IMP (-2, -8, -19, -20, -24) Carbapenem-resistant enterococci test method using real-time PCR, characterized in that the label is labeled at one end of at least one probe selected from the group consisting of a probe consisting of. 5. The method of claim 4,
A method for testing carbapenem-resistant enterobacteriaceae using real-time PCR, characterized in that at the same time using two probes labeled with a label that can be detected at different wavelengths. The method of claim 5,
Real-time PCR is characterized by detecting two target genes specific for carbapenem-resistant enterobacteria in one test tube at the same time using two probes labeled with a label that can be detected at different wavelengths. Method of testing carbapenem-resistant enterobacteriaceae using. Primer pairs of SEQ ID NOS: 1 and 2 for KPC genes specific for carbapenem-resistant enterococci, primer pairs of SEQ ID NOs: 3 and 4 for VIM genes specific for carbapenem-resistant enterobacteria, and NDM genes specific for carbapenem-resistant enterobacteria Primer pairs of SEQ ID NOs: 5 and 6, primer pairs of SEQ ID NOs: 7 and 8 for GES genes specific for carbapenem-resistant enterobacterial, IMP (-1, -3, -6, -10 Primer pairs of SEQ ID NOs: 9 and 10 for genes, IMP (-4, -26) specific for carbapenem-resistant enterobacteria, and IMPs specific for carbapenem-resistant enterococci. At least one primer pair selected from the group consisting of primer pairs of SEQ ID NOs: 13 and 14 for the gene of -2, -8, -19, -20, -24, DNA polymerase, dNTPs, PCR buffer and PCR Label of amplification product A real-time PCR amplification kit for detecting carbapenem-resistant enterobacteria, including vagina. The method of claim 7, wherein
The labeling substance is a probe that specifically binds to the KPC gene specific for carbapenem-resistant enterobacteria, an oligonucleotide having a nucleotide sequence of SEQ ID NO: 15 or a probe consisting of an oligonucleotide having a nucleotide sequence complementary to such oligonucleotide, carbapenem A probe specifically binding to a VIM gene specific for resistant enterobacteria, comprising a oligonucleotide having a nucleotide sequence of SEQ ID NO: 16 or an oligonucleotide having a nucleotide sequence complementary to such oligonucleotide, a NDM specific for carbapenem resistant enterobacteria A probe specifically binding to a gene, the probe consisting of an oligonucleotide having a nucleotide sequence of SEQ ID NO: 17 or an oligonucleotide having a nucleotide sequence complementary to such an oligonucleotide, carbapenem-resistant intestine A probe that specifically binds to a GES gene specific to a bacterium and comprises an oligonucleotide having a nucleotide sequence of SEQ ID NO: 18 or an oligonucleotide having a nucleotide sequence complementary to such an oligonucleotide, and an IMP specific to carbapenem-resistant enterobacteria ( -1, -3, -6, -10) A probe specifically binding to a gene, a probe consisting of an oligonucleotide having a nucleotide sequence of SEQ ID NO: 19 or an oligonucleotide having a nucleotide sequence complementary to such oligonucleotide, carbapenem A probe specifically binding to an IMP (-4, -26) gene specific for resistant enterobacteria, comprising a oligonucleotide having a nucleotide sequence of SEQ ID NO: 20 or an oligonucleotide having a nucleotide sequence complementary to such oligonucleotide, and Specific to Carbapenem-resistant Enterobacteriaceae Oligonucleotide having a nucleotide sequence of SEQ ID NO: 21 or an oligonucleotide having a nucleotide sequence complementary to such an oligonucleotide as a probe that specifically binds to a gene of IMP (-2, -8, -19, -20, -24) A real-time PCR amplification kit for detecting carbapenem-resistant enterobacteria, characterized in that it is labeled at one end of at least one probe selected from the group consisting of a probe consisting of. 9. The method of claim 8,
A real-time PCR amplification kit for detecting carbapenem-resistant enterobacteria, comprising simultaneously two probes labeled with a label that can be detected at different wavelengths.

Images